This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cephalosporins are widely used antibiotics, synthesized from 7-amino cephaslosporanic acid which is obtained by chemical deacylation of the natural antibiotic cephalosporin C. The process for this synthesis is expensive and results in the production of toxic wastes that are difficult to dispose of. In an attempt to increase the specificity for cephalosporin C by glutaryl-7-ACA acylase laboratory evolution has been used to produce a biocatalyst for the direct deacylation of cephalosporin C. Cephalosporin antibiotics account for 29% of the market share of anti-infective agents (approximately $7billion). Of great interest is an easy, effective and environmentally safe method to produce these antibiotics. In collaboration with L. Pollegioni (University of Insubria, Italy) we are pursuing structural studies of an of evolved glutaryl-7-amino cephalosporanic acid acylase as a protein scaffold with increased substrate specificity for cephalosporin. A mutant form of this acylase has been found with 100-fold increased activity on cephalosporin C as compared to glutaryl-7-amino cephalosporanic acid.